Synaptic plasticity has been suggested to be a cellular counterpart for learning and memory. However, it is practically impossible to visually monitor synapses actually undergoing synaptic plasticity at a given memory paradigm in a given neuronal network. This project proposal, written in response to the program announcement "Developing Novel Genetic Methods for Mapping Functional Neuronal Circuits and Synaptic Change", describes the development of a technology for visualizing synapses that are undergoing synaptic plasticity in neurons in living animal. This goal will be accomplished by a combination of two technologies: fluorescence resonance energy transfer (FRET) and two-photon laser scanning microscopy. We will first develop a FRET-based construct for optically measuring the CaMKII activity and actin polymerization/depolymerization equilibrium, which will allow non-destructive optical detection of CaMKII activation in intact neurons. Since the enzymatic activity of CaMKII is constitutively enhanced after the induction of NMDA receptor-mediated synaptic plasticity and this activity is required for maintaining synaptic potentiation, we expect that the activation of CaMKII will be a good indicator of synaptic plasticity. In contrast, in a work which we published, we found actin polymerization/depolymerization equilibrium can be detected with FRET and that it follows LTP and LTD respectively. To detect FRET at the synaptic structure, we will take advantage of a two-photon microscope. We will then test the feasibility of our strategy by expressing the construct in neurons and induce synaptic plasticity by either high-speed ionophoretic stimulation of individual synapses or local electrical stimulation or combined with detection of structural plasticity. Finally, we will generate a transgenic animal expressing this construct. The barrel cortex of the resultant animal will be observed with the two-photon microscope. Paradigms known to induce synaptic plasticity in this structure such as sensory deprivation will be tested to see whether synaptic plasticity is reflected by FRET. In summary, our technology will provide a unique system for detecting NMDAR-mediated synaptic plasticity with spatial resolution at the single synapse level on a sub-second time scale. This technique, in combination with technologies currently under development in other laboratories, such as in vivo two-photon imaging in freely moving animals and deep-structure two-photon microscope imaging with a relay lens, will pro- vide a versatile system for monitoring synaptic plasticity that cannot be achieved with existing experimental systems. In the future, this technique could be applied to higher mammals, such as macaque monkeys, after viral or transgenic introduction of our reporter construct. It may be possible to have monkeys perform a task and observe synaptic plasticity in the brain during learning of the task. [unreadable] [unreadable]